•A compact and sensitive biosensor based on an E-SMS optical fiber structure.•A label-free biosensor for the rapid detection of tuberculosis Ag85B protein.•These biosensors are cost-effective, simple to construct and easy to implement.•Biosensor detects interactions up to 80 ng/mL with sensitivity of 2.3 nm/(µg/ml).•Biosensor provided high selectivity in detection of the antigen, comparable to ELISA. Tuberculosis, caused by Mycobacterium tuberculosis bacillus, is one of the most prevalent, widespread, infectious, and deadliest diseases in the world. In this work, a compact and sensitive biosensor based on an etched single-mode–multimode–single-mode (E-SMS) optical fiber structure was developed for the rapid detection of Mycobacterium tuberculosis Ag85B protein in phosphate buffered saline solution. This biosensor, based on the multimode interference produced into an E-SMS structure, was fabricated by chemical etching of the cladding region of the multimode fiber, followed by the immobilization of the anti-Ag85B antibody on the surface of the etched region. The functionalized E-SMS biosensor provided high selectivity in the detection of Ag85B protein, with the ability to detect biological interactions in a range of 1.31 µg/mL to 4.36 µg/mL, with a sensitivity of 2.3 nm/(µg/mL); also, signal was even obtained at concentrations as low as 80 ng/mL. This sensitive label-free biosensing technology could contribute significantly improve Mycobacterium tuberculosis detection, management in clinical diagnosis, and medical treatment of infected patients.